The field of this invention is monooxygenase assays.
Monooxygenase include the numerous isoforms of the cytochrome P-450 enzymes. Because of the importance of these enzymes, particularly their activity in the liver, there is substantial interest in being able to assay for their activity and identify compounds that can modulate that activity. These enzymes serve to clear the blood of foreign factors. Unfortunately, in many cases these foreign factors are drugs, whose half-life is substantially diminished by virtue of being processed by the P-450 enzymes into inactive products. Also, reduced or modified activity of the P-450 enzymes may lead to poisoning or sensitivity to various agents, which in the normal person would be rapidly detoxified. The enzyme preparations are very expensive and each enzyme has multiple binding sites. Thus, multiple assays have to be performed to screen the enzyme(s).
With the advent of nanotechnology, there is an increased ability to perform numerous chemical and physical operations with very small volumes. This opportunity comes with the requirement that determinations have enhanced sensitivity to detect the fewer molecules that are present to provide the detectable signal. Part of the increased sensitivity may come from more sensitive detectors, but these are usually more expensive and are not readily available in most laboratories. The other opportunity is to provide assays that are more efficient in providing for detectable products, uses compounds that are readily accepted by the enzymes as substrates, and provide products with a strong signal, for fluorescent compounds, a high emission efficiency.
There is, therefore, substantial interest in providing P-450 assays that are rapid, accurate and can be performed in small volumes with low levels of enzyme and expensive reagents.
U.S. Pat. No. 5,179,013 and references cited therein describe assaying for novel cytochrome P-450 enyzmes. Assays for P-450 enzymes are also described in Schwaneberg, et al., Anal Biochem 1999, 1:269:359-66; Tremblay, et al., Anal Biochem 1999, 276:215-26; Jansen, et al., J Chromatogr B Biomed Appl 1996, 684:133-45 and Eguchi et al., Xenobiotica 1996, 26:755-63. Other references that may be of interest include Hartmann and Frotscher, Arch Pharm 1999, 332:358-62; Ubeaud, et al., Eur J. Pharm Sci 1999, 8:255-60; Ertl, et al., Toxicol Appl Pharmcol 1999, 157:157-65; and Sanderson, et al., Toxicol Appl Pharmacol 1996, 137:316-25.
Methods and compositions are provided for determinations of monooxygenase enzymes, particularly P-450 enzymes, using a substrate having an ether group comprising an oxidizable xcex1-hydrogen, resulting in an aldehyde product. The aldehyde is reacted in situ with a fluorescent hydrazine and the resulting reaction mixture separated by capillary electrophoresis. The hydrazone product indicates the reaction occurrence and the area under the peak may be used for quantitation. Kits can be provided for performing the assay.